Project Summary/Abstract It is crucial to better understand immune evasion mechanisms in head and neck cancers in order to enhance their susceptibility to immunotherapy. About 90% of head and neck cancers are squamous cell carcinomas (HNSCC). Recurrent or metastatic HNSCCs are being treated with checkpoint blockade immunotherapy targeting programmed death 1 (PD-1), a co-inhibitory receptor on T cells. However, only a subset of HNSCC patients responded to such anti-PD-1 therapy (10-20%). Thus, there is an urgent need to elucidate mechanisms underlying therapy unresponsiveness to single blockade of PD-1. Apart from PD-1, T cells express other co-inhibitory receptors that can also induce immunosuppressive phenotypes, such as lymphocyte activation gene-3 (LAG-3). However, the role of such receptors remains poorly defined in immune evasion of HNSCCs (e.g., LAG-3). Our preliminary data show that HNSCC patients exhibit a highly heterogeneous pattern of tumor infiltrating lymphocytes (TILs); however, the molecular drivers underlying such differential immune phenotypes remain largely unknown. Completion of our proposed studies may generate novel insight into the mechanisms that determine the success or failure of checkpoint blockade immunotherapy. We expect our studies to delineate the comprehensive immune landscape of HNSCCs in human patients. The knowledge gained would provide critical steps toward improving immunotherapy by targeting additional co-inhibitory receptors with a more rational design and overcoming the dysfunctional progression of TILs. Our long-term goal is to elucidate immune evasion mechanism and improve therapeutic strategies of HNSCCs. HNSCC development often associates with oncogenic mutations, such as heterozygous loss of Smad4, gain-of-function mutations of PIK3CA or loss-of-function mutations of Notch1. It remains largely unknown how HNSCCs evade immune recognition. To address this question, we performed studies with a transplanted SCC model caused by combining KrasG12D mutation and Smad4 loss (termed KRS-SCC). We found that KRS-SCC tumors completely escaped T cell-mediated anti-tumor responses, manifested with exhausted CD8 and CD4 TILs co-expressing PD-1 and LAG-3. Consistently, dual inhibition of PD-1 and LAG-3 suppressed the growth of KRS-SCCs. We propose to employ our unique mouse models and human patient samples to further elucidate immune evasion mechanisms of HNSCCs. Our proposed studies may substantially advance our understanding in mechanisms that underlie therapy failure of single PD-1 blockade. Relevance to public health. We envision that our studies will provide substantial advances in understanding the mechanisms that underlie therapy failure of PD-1 blockade in HNSCCs. We anticipate that our proposed studies will reveal the connection between intrinsic characteristics of tumor cells and immune signature of TILs. These studies not only address fundamental questions in cancer immunology but also lay a scientific foundation for developing novel therapy of HNSCCs in the targeted patient populations with a more rationalized design.